The invention concerns a method for the rapid, simultaneous isolation of genomic desoxyribonucleic acid (DNA) and cellular total ribonucleic acid (RNA) from various starting materials.
It is of great importance for a multitude of laboratories working in the fields of biology, molecular biology, medical analyses and biochemistry. Thus, the fields of application are molecular biology, biochemistry, gene technology, medicine, veterinary medicine and all related fields.
The simultaneous isolation of genomic DNA and cellular total RNA from one and the same starting material has till the present day been bound only to a few less practicable methods. Thus, Raha, S., Merante, F., Proteau, G. and Reed, J. K. (GATA, 1990, 7 (7): 173-177) describe a method for the separation of genomic DNA and cellular total RNA through selective precipitation steps using lithium chloride. A further possibility of isolating DNA and RNA simultaneously is based on an ultracentrifugation through a caesium chloride gradient for pelleting RNA and dialysing subsequently DNA from the guanidine phase (Coombs, L. M., Pigott, D.; Proctor, A., Eydmann, M., Denner, J. and Knowles, M. A.; Anal. Biochem. (1990); 188; 338-343). Such a method is very time-consuming (at least 48 hours) and requires remarkable expenditure of apparatuses (ultra-sound centrifugation equipment, special rotors).
A method, for the time being, frequently used and also commercially available is based on the use of a reagent consisting of guanidine thiosulphate and phenol. The biological material is homogenised in this reagent with RNA reaching an aqueous phase after adding chloroform and separating the phases and being precipitated from it. The remaining interphase or the phenolic phase contains proteins as well as genomic DNA. By modifying the pH and a repeated separation of phases the genomic DNA is also to be converted into the aqueous phase and again precipitated from it (Chomczynski, P., Biotechniques 1993, 15(3): 532-536).
In principle, according to the state of the art you have to proceed on the fact that isolated cellular total RNA is contaminated by genomic DNA.
Thus, the aqueous phase obtained by means of the reagent developed and used by Chomcynski contains in addition to RNA also genomic DNA which is then also precipitated from this phase thus being contained as a contaminating component in the final RNA preparation. Notably the contamination of isolated cellular RNA by genomic DNA appears to be a grave problem for a multitude of further applications of RNA. Thus, e.g. the application of an RNA protection assay is necessarily bound to a RNA free from DNA. Furthermore, the RNA used for a multitude of RT-PCR reactions has to be free from a contamination by genomic RNA. Thus, there is no possibility of detecting whether the PCR fragment resulting from the contaminating DNA or from RNA was amplified e.g. in investigations of expression of cDNA constructions in transgenic organisms and also in the detection of the expression of intronless genes and also of still unknown gene sequences. Amplificates derived from the genomic DNA and from a RNA would be of the same length. In addition, also a number of further molecular biological methods such as e.g. DDRT-PCR or cell-free protein biosyntheses in the form of coupled in-vitro transcriptions/translation systems depend on a RNA preparation free from DNA.
This shows the importance of isolating total RNA free from genomic DNA. A further problem consists in the duration of the preparation to simultaneously isolate genomic DNA and cellular total RNA and the work connected with it.
The only isolation system commercially available, for the time being, takes at least 3 hours to carry out the simultaneous isolation of the two nucleic acid fractions and requires a quite remarkable number of reactions vessels and fine chemicals. Furthermore, when applying all these methods a comparatively big quantity of biological starting materials is necessary. Thus, mostly a simultaneous isolation of the two nucleic acids will be no longer possible if only limited quantities of materials for investigation will be available.